DM2 subjects with DKD were recruited at the Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo. Healthy control individuals matched by age and gender were selected at Faculdade de Medicina da Universidade de São Paulo. All participants were properly informed about the procedures and the study and signed an informed written consent form that was previously approved by The Ethical Committee for Human Research Protocols of the Clinical Hospital (#15024), in accordance with the Declaration of Helsinki.
Subjects with other chronic diseases, rapid loss in GFR (> 3 mL / min / year), refractory hypertension, BMI < 18.5 Kg / m2, current smoke or alcohol abuse were not included. Blood was drawn after overnight fasting and plasma was immediately isolated in refrigerated centrifuge (4°C). Glycemia, triglycerides (TG), total cholesterol (TC), HDL cholesterol (HDLc), fructosamine, TSH, creatinine and urea were determined in plasma by enzymatic techniques after overnight fasting and albumin in 24h-urine. HbA1c was determined by high performance liquid chromatography (HPLC). DKD individuals were categoried according to the GFR above 60 mL/min/1.73 m2 plus AER stages A1 (< 30 mg/g creatinine) and A2 (30 - 300 mg/g creatinine) and GFR below 60 mL/min/1.73 m2 plus stage A3 (> 300 mg/g creatinine). Control subjects presented GFR above 60 mL/min/1.73 m2 plus A1.
Isolation of lipoproteins
Venous blood samples were drawn after overnight fasting and plasma immediately isolated in a refrigerated centrifuge. Preservatives were added to the plasma and density adjusted with bromide potassium to 1.21 g/mL. Low- density (LDL; d = 1.019-1.063 g/mL) and high- density lipoprotein (HDL; d = 1.063-1.21 g/mL) were isolated from plasma by discontinous density gradient ultracentrifugation (100 000g, 24 h, 4 °C, Sw40 rotor; Beckman ultracentrifuge). Samples were dialyzed against phosphate buffer saline containing EDTA (PBS).
HDL composition in lipids and apoA- I
The amount of lipids and apo A-I in HDL were determined, respectively, by enzymatic techniques [TC and TG; Labtest diagnóstica S. A., Minas Gerais, Brazil; phospholipids (PL); Randox Laboratorier LTD. Crumlin, Co. Antrem, United Kingdom] and immunoturbidimetry.
Determination of total AGE and pentosidine in HDL
The contents of total AGE and pentosidine were determined by fluorescence measurement (Synergy HT Multi-Mode Microplate Reader, SpectraMax M5). Samples were excited at a wavelength of 370nm and the fluorescence emitted at 440 nm and 378 nm, respectively, for total AGE and pentosidine.
Proteolytic digestion of HDL
The HDL protein concentration was determined by the Bradford assay (Bio-Rad, Hercules, CA, USA). Ten micrograms of HDL-protein was solubilized in 100 mM ammonium bicarbonate, dithiothreitol, and iodoacetamide, following digestion with trypsin (1:40, w/w Promega, Madison, WI,USA) for 4 h at 37 °C. Trypsin was further added to samples (1:50, w/w HDL) and the incubation was done overnight at 37 °C. Samples were desalted using solid phase extraction (Oasis PRIME HLB SPE column; Waters) after acidic hydrolysis with 2% trifluoroacetic acid and kept frozen at - 80°C until MS analyses. Prior to MS analysis, samples were resuspended in 0.1% formic acid (final protein concentration of 25 ng/μL).
Targeted proteomic analyses
Digested HDL proteins (50 ng protein) were quantified by parallel reaction monitoring (PRM), as previously described [12]. Briefly, an Easy-nLC 1200 UHPLC (Thermo Scientific, Bremen, Germany) was used for peptide separation. Acquisition of the data was performed in an Orbitrap Fusion Lumos mass spectrometer (Thermo Scientific, Bremen, Germany) using a nanospray Flex NG ion source (Thermo Scientific, Bremen, Germany). A scheduled (3-min window) inclusion list containing m/z of precursor peptides of interest and corresponding retention times was generated using Skyline software [13].
Selection of HDL peptides for targeted quantification
PRM methodology was assembled from shotgun proteomics analyses as previously described [12]. Ninety-one proteins were identified, but this number was reduced to 47 proteins eliminating potentials contaminant proteins (keratin, proteins with <2 unique peptides and peptides with high interfering signal and mass error >10 ppm). Peptides susceptible to ex-vivo modification (e.g., methionine-containing peptides) were also avoided, and only peptides satisfactorily detected were included in the final analysis. After our exclusion criteria, 31 proteins remained. For statistical analysis, we considered the best peptide that represents each protein of interest. In order to find these surrogate peptides, firstly the peptide pair with best Pearson’s correlation coefficient was determined. From these 2 peptides, the peptide with the lowest CV was finally selected. The 31 surrogate peptides chosen for HDL proteins are highlighted in Supplementary Table 1.
Acetylation of LDL
LDL was acetylated as previously described by Basu et al [14]. Samples were extensively dialyzed before incubation with macrophages.
Measurement of 14C-cholesterol efflux
Bone marrow-derived cells were isolated from mice and macrophages were differentiated as previously described [15]. Cells were overloaded with acetylated LDL (50 µg/mL DMEM) and 14C-cholesterol (0.3 µCi/mL) for 48 h. HDL from controls and DKD subjects (50 µg/mL) were utilized as cholesterol acceptors in 6- h incubations and cholesterol efflux calculated as previous described [8].
Measurement of the HDL antioxidant activity
The ability of HDL from controls and DKD individuals in inhibiting LDL oxidation was determined by incubation of LDL (40 µg/mL) isolated from a unique healthy plasma donor with CuSO4 solution (1 mL) in the presence of HDL (80 µg/mL). Lipoproteins were dialyzed against PBS without EDTA prior incubations. The absorbance at 234 nm was continuously monitored every 3 min during 4 h and the lag time phase for LDL oxidation (min) and the maximum ratio of conjugated dienes formation calculated [15].
Measurement of the HDL antiinflammatory activity
BMDM were isolated and cultured as previously described; then overloaded with acetylated LDL (50 µg/mL DMEM) and treated for 24 h with HDL from controls and DKD subjects. After washing, macrophages were incubated with lipopolyssacharide (LPS; 1 µg/mL DMEM) for 24 h. Medium was collected and the amount of TNF alpha and interleukin 6 (IL-6) determined by ELISA (R&D System-Duo Set, Minneapolis, EUA) [9].
Statistical analysis
Statistical analysis was performed using the GraphPad Prism 5 program (GraphPad Software, Inc. 2007). Comparisons were made by one-way analysis of variance (ANOVA) with Dunnett post-test, Student t test and Sperman linear correlation as appropriated. The value of p < 0.05 was considered statistically significant. Proteome data were compared by multiple comparisons.